Heteromultimeric proteins and methods of use thereof

Abstract

The present application provides heteromultimeric proteins, such as bispecific antibodies, comprising a first antibody heavy chain constant domain 3 (CH3)-containing polypeptide having a substitution relative to a wildtype CH3 domain at amino acid position 354 with a bulky hydrophobic amino acid, and/or a second CH3-containing polypeptide comprising a substitution relative to a wildtype CH3 domain at ammo acid position 347 with a negatively charged amino acid residue. Also provided are polypeptides, nucleic acids and vectors encoding such polypeptides, pharmaceutical compositions, methods of preparation and methods of treatment using the heteromultimeric proteins.

What is claimed is: 
     
         1 . A heteromultimeric protein comprising a first polypeptide comprising a first IgG heavy chain constant domain 3 (CH3) domain and a second polypeptide comprising a second IgG CH3 domain, wherein the first IgG CH3 domain comprises a substitution relative to a wildtype IgG CH3 domain at position 354 with an aromatic hydrophobic amino acid, and the second IgG CH3 domain comprises a substitution relative to a wildtype IgG CH3 domain at amino acid position 347 with a negatively charged amino acid, wherein the first IgG CH3 domain comprises a lysine (K) at amino acid position 360, and wherein the amino acid residue numbering is based on EU numbering. 
     
     
         2 . The heteromultimeric protein of  claim 1 , wherein the second IgG CH3 domain comprises a tyrosine (Y) at amino acid position 349. 
     
     
         3 . The heteromultimeric protein of  claim 1 , wherein the first IgG CH3 domain comprises a substitution selected from the group consisting of S354Y, S354F and S354W. 
     
     
         4 . The heteromultimeric protein of  claim 1 , wherein the second IgG CH3 domain comprises a substitution selected from the group consisting of Q347E and Q347D. 
     
     
         5 . The heteromultimeric protein of  claim 1 , wherein the first IgG CH3 domain and the second IgG CH3 domain further comprise knob-into-hole residues. 
     
     
         6 . The heteromultimeric protein of  claim 1 , wherein the first polypeptide is an antibody heavy chain, and the second polypeptide is an antibody heavy chain. 
     
     
         7 . The heteromultimeric protein of  claim 6 , wherein the heteromultimeric protein comprises one or more antibody light chains. 
     
     
         8 . The heteromultimeric protein of  claim 7 , comprising:
 (a) a first heavy chain comprising from the N-terminus to the C-terminus: a first heavy chain variable domain (VH1), a first heavy chain constant domain 1 (CH1), a first heavy chain constant domain 2 (CH2), and the first CH3 domain;   (b) a first light chain comprising from the N-terminus to the C-terminus: a first light chain variable domain (VL1), and a first light chain constant domain (CL);   (c) a second heavy chain comprising from the N-terminus to the C-terminus: a second heavy chain variable domain (VH2), a second CH1, a second CH2, and the second CH3 domain; and   (d) a second light chain comprising from the N-terminus to the C-terminus: a second light chain variable domain (VL2), and a second CL;   wherein VH1 and VL1 associate to form a first antigen binding site that specifically binds to a first target, and VH2 and VL2 associate to form a second antigen binding site that specifically binds to a second target.   
     
     
         9 . The heteromultimeric protein of  claim 8 , wherein the first antigen binding site specifically binds a tumor antigen and the second antigen binding site specifically binds CD3. 
     
     
         10 . The heteromultimeric protein of  claim 8 , comprising:
 (a) a first heavy chain comprising from the N-terminus to the C-terminus: a third heavy chain variable domain (VH3), a third CH1, the VH1, the first CH1, the first CH2, and the first CH3 domain;   (b) a first light chain comprising from the N-terminus to the C-terminus: a third light chain variable domain VL3, a third CL, the VL1, and the first CL;   wherein VH3 and VL3 associate to form a third antigen binding site that specifically binds to a third target.   
     
     
         11 . The heteromultimeric protein of  claim 7 , comprising:
 (a) a first heavy chain comprising from the N-terminus to the C-terminus: a first VHH, a first heavy chain constant domain 2 (CH2), and the first CH3 domain;   (b) a second heavy chain comprising from the N-terminus to the C-terminus: a first heavy chain variable domain (VH1), a second CH1, a second CH2, and the second CH3 domain; and   (c) a light chain comprising from the N-terminus to the C-terminus: a first light chain variable domain (VL1), and a first CL;   wherein VH1 and VL1 associate to form a first antigen binding site that specifically binds to a first target, and the first VHH specifically binds to a second target.   
     
     
         12 . The heteromultimeric protein of  claim 1 , wherein the heteromultimeric protein is an immunoadhesin or an antibody-immunoadhesin chimera. 
     
     
         13 . One or more nucleic acid(s) encoding the heteromultimeric protein of  claim 1 . 
     
     
         14 . A vector comprising the one or more nucleic acid(s) of  claim 13 . 
     
     
         15 . A host cell comprising the one or more nucleic acid(s) of  claim 13 . 
     
     
         16 . A method for preparing a multispecific antibody or a heteromultimeric protein, comprising:
 (a) culturing the host cell of  claim 15  under conditions that allow expression of the one or more nucleic acid(s) or vector; and   (b) recovering the multispecific antibody or the heteromultimeric protein from the host cell culture.   
     
     
         17 . A pharmaceutical composition comprising the heteromultimeric protein of  claim 1 , and a pharmaceutically acceptable excipient. 
     
     
         18 . A method for treating a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 17 . 
     
     
         19 . A method of generating a heteromultimeric protein that specifically binds to a first target and a second target, comprising:
 (a) providing a first polypeptide comprising a first binding domain that specifically binds to the first target, and a first IgG CH3 domain; and   (b) providing a second polypeptide comprising a second binding domain that specifically binds to the second target, and a second IgG CH3 domain;
 wherein:
 (i) the first IgG CH 3  domain comprises a substitution relative to a wildtype IgG CH3 domain at position 354 with an aromatic hydrophobic amino acid, wherein the first IgG CH3 domain comprises a lysine (K) at amino acid position 360, and the second IgG CH3 domain comprises a substitution relative to a wildtype IgG CH3 domain at amino acid position 347 with a negatively charged amino acid; or 
 (ii) the first IgG CH 3  domain comprises a substitution relative to a wildtype IgG CH3 domain at position 347 with a negatively charged amino acid, and the second IgG CH3 domain comprises a substitution relative to a wildtype IgG CH3 domain at amino acid position 354 with an aromatic hydrophobic amino acid, wherein the second IgG CH3 domain comprises a lysine (K) at amino acid position 360; 
 
 and wherein the amino acid residue numbering is based on EU numbering.